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Imiterere y’amara y’umuntu ishyiraho imiterere ya crypt-villus ya microarchitecture ya 3D epithelial n’imiterere y’ahantu. Iyi miterere idasanzwe ikenewe kugira ngo ikomeze homeostasis y’amara irinda niche y’uturemangingo tw’inyuma mu gice cy’imbere cy’inyuma ... Kugaragaza epithelia ya 3D yashyizweho hakoreshejwe uburyo bwinshi bwo gufata amashusho. Iyi protocole igera ku kuvugurura microarchitecture y'imikorere y'amara binyuze mu kugenzura urujya n'uruza rw'amazi mu minsi 5. Uburyo bwacu bwo gukora morphogenesis muri vitro bukoresha stress ijyanye na physiologically hamwe n'ingendo za mekanike kandi ntibusaba ubuhanga buhambaye cyangwa uburyo bwo guhinduranya utunyangingo, bishobora kurusha ubundi buryo busanzweho. Turatekereza ko protocole yacu ishobora kugira ingaruka nini ku muryango w'ubushakashatsi ku buvuzi, itanga uburyo bwo kuvugurura imiterere y'amara ya 3D muri vitro mu bijyanye n'ubuvuzi, ubuvuzi, n'imiti.
Ubushakashatsi bwerekanye ko uturemangingo twa Caco-2 two mu mara twakuriye mu bikoresho bya "gut-on-a-chip"1, 2, 3, 4, 5 cyangwa "bilayer microfluidic devices"6, 7 dushobora kunyura mu buryo bwa "3D morphogenesis" mu buryo bwikora mu buryo bwa "vitro" tudasobanukiwe neza uburyo bw'ibanze. Mu bushakashatsi bwacu buherutse gukorwa, twasanze gukuraho utunyangingo twa "morphogen antagonists" twakuwe mu bikoresho bya "culture" ari ngombwa kandi bihagije kugira ngo habeho "3D epithelial morphogenesis" mu buryo bwa "vitro", nk'uko byagaragajwe na Caco-2 na organoids z'amara zikomoka ku barwayi. Uturemangingo tw’ingirabuzimafatizo twemewe. Muri ubu bushakashatsi, twibanze cyane cyane ku ikorwa ry’uturemangingo n’ikwirakwizwa ry’uturemangingo tw’umurwayi ukomeye wa Wnt, Dickkopf-1 (DKK-1), mu turemangingo tw’ingirabuzimafatizo n’ibikoresho bya microfluidic byahinduwe birimo Transwell inserts, byitwa "Hybrid Chip". Tugaragaza ko kongeramo uturemangingo tw’ingirabuzimafatizo twa Wnt (nka DKK-1, Wnt repressor 1, poroteyine 1 ifitanye isano na frizzled, cyangwa Soggy-1) mu turemangingo tw’ingirabuzimafatizo bibuza imiterere cyangwa bigahungabanya urwego rwa 3D epithelial, bigaragaza ko stress irwanya mu gihe cyo kwita ku miterere y’amara ari yo ituma amara ahinduka muri vitro. Kubwibyo, uburyo bufatika bwo kugera ku miterere ikomeye ku gice cy’ingirabuzimafatizo ni ugukuraho cyangwa kubungabunga urwego rw’uturemangingo tw’ingirabuzimafatizo twa Wnt mu gice cy’ingirabuzimafatizo binyuze mu gusukura (urugero, mu turemangingo tw’ingirabuzimafatizo cyangwa ikoranabuhanga rya hybrid-on-a-chip) cyangwa gukwirakwiza. Ingirabuzimafatizo z’ingirabuzimafatizo (urugero, zituruka kuri Transwell zinjira mu bigega binini by’ingirabuzimafatizo muri amariba).
Muri iyi porotoli, dutanga uburyo burambuye bwo gukora ibikoresho bya "gut-on-a-chip" hamwe na "Transwell-insertable hybrid chips" (intambwe ya 1-5) kugira ngo hakorwe uturemangingo twa "intestine" duto duto ku turemangingo duto dushingiye kuri polydimethylsiloxane (PDMS) (intambwe ya 6A, 7A, 8, 9) cyangwa uturemangingo twa polyester twa Transwell inserts (intambwe ya 6B, 7B, 8, 9) kandi hakorwe "morphogenesis" ya 3D muri "vitro" (intambwe ya 10). Twanagaragaje imiterere y'uturemangingo n'uturemangingo bigaragaza "histogenesis" yihariye y'uturemangingo no gutandukanya uturemangingo dushingiye ku murongo dukoresheje uburyo bwinshi bwo gushushanya (intambwe ya 11-24). Dukora "morphogenesis" dukoresheje uturemangingo twa "intestine" duto duto, nka Caco-2 cyangwa "organoids" mu mara, mu buryo bubiri bwo gukora "culture" hamwe n'ibisobanuro bya tekiniki birimo guhindura ubuso bw'uturemangingo duto, gukora "monolayers" za 2D, na "biochemical" mu mara no kongera gukora "biomechanical microenvironment". muri "vitro". Kugira ngo hakorwe "morphogenesis" ya 3D iturutse kuri "2D epithelial". mu buryo bumwe, twakuyeho ibintu byombi byatewe n’imiterere y’ibinyabuzima binyuze mu gushyira mu gice cy’imbere cy’umuco. Amaherezo, dutanga ishusho y’akamaro k’urwego rw’inyuma rw’umubiri rushobora kuvugururwa rwa 3D rushobora gukoreshwa mu kwerekana imikurire y’inyuma y’umubiri, imiterere y’ingirabuzimafatizo z’ibinyabuzima, kwandura indwara ziterwa n’udukoko, imvune ziterwa n’ubushyuhe, imikorere mibi y’ingirabuzimafatizo z’umubiri, n’uburyo bwo kuvura bushingiye kuri probiotic.
Protocole yacu ishobora kuba ingirakamaro ku bahanga benshi mu bya siyansi mu by’ibanze (urugero, ibinyabuzima by’amara, ibinyabuzima by’uturemangingo fatizo, n’ibinyabuzima by’iterambere) n’ubushakashatsi bukoreshwa (urugero, gupima imiti mbere yo kuvura, kwerekana indwara, ubuhanga mu by’imitsi, na gastroenterology) ingaruka zagutse. Kubera ko protocole yacu ishobora kongera kubyara no gukomera mu gutera imiterere ya 3D y’impinduka mu mara muri vitro, dutekereza ko ingamba zacu za tekiniki zishobora gukwirakwizwa ku bantu biga imiterere y’ibimenyetso by’uturemangingo mu gihe cyo gukura kw’amara, kongera kuvugurura cyangwa kongera kumera neza. Byongeye kandi, protocole yacu ni ingirakamaro mu gusuzuma indwara hifashishijwe imiti itandukanye yandura nka Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9 cyangwa Vibrio cholerae. Abareba indwara n'inkomoko yazo nabyo ni ingirakamaro. Gukoresha sisitemu ya microphysiology y'igifu ishobora gutuma habaho ubwiyongere bw'indwara mu gihe kirekire (long-chip gut microphysiology) 10 no gusuzuma ubwirinzi bw'umuntu, uburyo ubudahangarwa bw'umubiri butera ndetse no gusana imvune ziterwa n'udukoko mu nzira y'igifu (GI) 11. Izindi ndwara za GI zifitanye isano n'indwara y'igifu iva, indwara ya celiac, indwara ya Crohn, indwara y'ibisebe, pouchitis, cyangwa indwara y'igifu ishobora kwiganwa iyo ibice bya epithelial by'amara bya 3D biteguwe hakoreshejwe ibice bya epithelial by'amara bya 3D by'umurwayi, izi ndwara zirimo kugabanuka kw'amagufwa, gufungana kw'amagufwa, kwangirika k'umuyoboro w'amaraso, cyangwa imbogamizi y'ingufu z'amara. Biopsy cyangwa stem cell-derived intestine organoids12,13. Kugira ngo bagaragaze neza imiterere y'indwara, abasomyi bashobora gutekereza kongeramo ubwoko bw'uturemangingo bujyanye n'indwara, nka cells mononuclear blood cells (PBMCs) z'umurwayi, ku buryo bukubiyemo microarchitectures za 3D intestine villus-crypt. uturemangingo tw'ubudahangarwa twihariye ku mubiri, 5.
Kubera ko imiterere mito ya epithelial ya 3D ishobora gukosorwa no kugaragazwa hatabayeho uburyo bwo gutandukanya, abareba bakora ku nyandiko z'ahantu hamwe n'ishusho ifite ubushobozi bwo hejuru cyangwa ifite ubushobozi bwo hejuru bashobora gushishikazwa no gushushanya imiterere y'ahantu na poroteyine ku bice by'imbere. Bashishikajwe n'ikoranabuhanga. Igisubizo ku mikorobe cyangwa ibintu bitera ubudahangarwa bw'umubiri. Byongeye kandi, ikiganiro 10, 14 cy'umukozi wo mu gihe kirekire gihuza imiyoboro y'amara gishobora gushyirwa mu gice cy'amara cya 3D binyuze mu gutera amoko atandukanye ya mikorobe, uduce twa mikorobe cyangwa udukoko two mu ifu, cyane cyane mu gice cy'amara. muri urwo rubuga. Ubu buryo bukurura cyane abantu biga ibijyanye no kwirinda indwara z’imitsi, indwara z’igifu, indwara z’imibiri y’abantu, ubuvuzi bw’imibiri n’ibinyabuzima bivura bashaka guhinga mikorobe z’amara zitari zarahinzwe muri laboratwari. Niba porotoli yacu ya in vitro morphogenesis ishobora guhindurwa kugira ngo ikoreshwe mu buryo bwo gukwirakwiza, nko gushyiramo imiti myinshi mu masahani 24, 96 cyangwa 384 y’amasunzu ahora yubaka ibice by’imbere, porotoli ishobora kandi gukwirakwizwa ku bakora imiti, imiti cyangwa uburyo bwo gusuzuma ibiribwa mu buryo bwo hejuru cyangwa bwo kwemeza inganda z’ibiribwa. Nk’igihamya cy’amahame, duherutse kwerekana ko bishoboka ko sisitemu ya multiplex high-throughput morphogenesis ishobora gukwirakwizwa ku masahani 24 y’amasunzu. Byongeye kandi, ibicuruzwa byinshi by’ingingo byashyizwe mu bucuruzi16,17,18. Kubwibyo, kwemeza uburyo bwacu bwa in vitro morphogenesis bushobora kwihutishwa kandi bugakoreshwa na laboratwari nyinshi z’ubushakashatsi, inganda cyangwa za leta n’inzego zishinzwe kugenzura kugira ngo basobanukirwe uburyo bwo kuvugurura imiterere y’uturemangingo tw’amara mu buryo bwo gupima imiti cyangwa Uburyo bwo kuvura indwara hakoreshejwe imiti (biotherapeutics) Gukoresha imiti mu buryo bwa 3D gupima no kuyitwara byapimwe hakoreshejwe uburyo bwa 3D gusimbura utundi dukoko cyangwa hakoreshejwe uburyo bwihariye cyangwa bw’ubucuruzi bwo gupima uburyo bwo kubyara ingingo mu mara kugira ngo harebwe uburyo bwo kongera kubyara uburyo bwo kubyara mu mara.
Umubare muto w'ingero z'igerageza zijyanye n'abantu zakoreshejwe mu kwiga imiterere y'ingirabuzimafatizo z'amara, ahanini bitewe no kutagira protocole zishobora gushyirwa mu bikorwa zo gutera imiterere y'ingirabuzimafatizo za 3D muri vitro. Mu by'ukuri, ubumenyi bwinshi buriho ubu ku miterere y'ingirabuzimafatizo bushingiye ku bushakashatsi ku nyamaswa (urugero, zebrafish20, imbeba21 cyangwa inkoko22). Ariko, zikora cyane kandi zihenze, zishobora kuba ziteye amakenga mu by'umuco, kandi icy'ingenzi kurushaho, ntizigena neza inzira z'iterambere ry'umuntu. Izi modele nazo zifite ubushobozi buke bwo kugeragezwa mu buryo bwinshi bushobora kwaguka. Kubwibyo, protocole yacu yo kuvugurura imiterere y'ingirabuzimafatizo za 3D muri vitro irusha imiterere y'inyamaswa muri vivo kimwe n'izindi modele gakondo zo guteza imbere ingirabuzimafatizo za 2D. Nkuko byasobanuwe mbere, gukoresha imiterere y'ingirabuzimafatizo za 3D byadufashije gusuzuma aho ingirabuzimafatizo zitandukanye ziri mu murongo wa crypt-villus mu gusubiza ibintu bitandukanye bya mucosal cyangwa ubudahangarwa bw'umubiri. Ibyiciro bya 3D muri epithelial bishobora gutanga umwanya wo kwiga uburyo ingirabuzimafatizo za mikorobe zihangana mu gukora ahantu hamwe n'imihindagurikire y'ibidukikije mu rwego rwo guhangana n'ibintu bizikikije (urugero, imbere ugereranyije uturemangingo tw’inyuma, gusohora IgA na peptides zirwanya mikorobe). Byongeye kandi, imiterere ya epithelial ya 3D ishobora kudufasha gusobanukirwa uburyo mikorobe zo mu nda zitunganya imiryango yazo kandi zigakora metabolites za mikorobe mu buryo bw’ubufatanye (urugero, aside fatty acids ngufi) zigize imiterere y’uturemangingo n’uturemangingo tw’uturemangingo mu miyoboro y’inyuma. Ibi bintu bishobora kugaragazwa gusa iyo uturemangingo twa 3D epithelial dushinzwe muri vitro.
Uretse uburyo bwacu bwo gukora imiterere y’uturemangingo tw’amara ya 3D, hari uburyo butandukanye bwo gukora muri vitro. Gutunganya organoid mu mara ni uburyo bugezweho bwo gukora ingirabuzimafatizo bushingiye ku buhinzi bw’uturemangingo tw’amara munsi y’imiterere yihariye ya morphogen23,24,25. Ariko, gukoresha moderi za organoid za 3D mu gusesengura ubwikorezi cyangwa co-cultures za host-microbiome bikunze kugorana kuko lumen y’amara iba iri muri organoid, bityo, kwinjira kw’ibice by’urumuri nk’uturemangingo twa mikorobe cyangwa antigens zo hanze ni bike. Kubona lumens za organoid bishobora kunozwa hakoreshejwe microinjector,26,27 ariko ubu buryo burakoreshwa cyane kandi busaba ubumenyi bwihariye kugira ngo bukore. Byongeye kandi, cultures gakondo za organoid ziguma mu duce twa hydrogel mu gihe cy’imimerere idahinduka ntizigaragaza neza biomekanike ikora mu mubiri.
Ubundi buryo bukoreshwa n'amatsinda menshi y'ubushakashatsi bukoresha ibyuma bya 3D hydrogel byateguwe mbere kugira ngo byigane imiterere y'igifu binyuze mu gukuriramo utunyangingo tw'amara y'umuntu twashyizwe ku buso bwa gel. Kora ibyuma bya hydrogel ukoresheje ibishushanyo bya 3D byacapwe, byakozwe mu buryo bwa micro-milled, cyangwa byakozwe na lithographically. Ubu buryo bwerekana uburyo uturemangingo twa epithelial twihariye twiyubatse muri vitro hamwe n'imiterere ya morphogen ijyanye n'imiterere y'umubiri, bigashyiraho imiterere ya epithelial ifite ikigereranyo cyo hejuru hamwe n'uburyo bwo guhuza stroma-epithelial binyuze mu gushyiramo uturemangingo twa stromal muri scaffold. Ariko, imiterere y'ibyuma byateguwe mbere ishobora kubuza kwerekana inzira ya morphogenetike ubwayo. Izi moderi kandi ntizitanga ubwiyongere bw'urumuri cyangwa interstitial, zidafite stress yo gukata amazi uturemangingo tw'amara dukeneye kugira ngo tugire imiterere ya morphogenesis kandi tugire imikorere ya physiological. Ubundi bushakashatsi buherutse bwakoresheje ibyuma bya hydrogel mu buryo bwa microfluidic hamwe n'imiterere ya epithelial y'amara hakoreshejwe uburyo bwa laser-etching. Organoids y'amara y'imbeba ikurikiza imiterere yakozwe kugira ngo ikore imiterere ya tubula y'amara, kandi amazi yo mu miringoti ashobora Gusubiramo hakoreshejwe module ya microfluidics. Ariko, iyi moderi ntabwo igaragaza inzira za morphogenetike zitunguranye cyangwa ngo zishyiremo ingendo za mechanobiological mu mara. Ubuhanga bwo gucapa 3D buturutse mu itsinda rimwe bwashoboye gukora imiyoboro mito y'igifu ifite inzira za morphogenetike zitunguranye. Nubwo hakozwe ibice bitandukanye by'igifu muri iyo moderi, iyi moderi kandi nta ruziga rw'amazi rugenda neza no guhinduka kwa mekanike. Byongeye kandi, ubushobozi bwo gukora moderi bushobora kuba buke, cyane cyane nyuma y'uko inzira ya bioprinting irangiye, bigahungabanya imiterere y'igerageza cyangwa imikoranire hagati y'uturemangingo n'uturemangingo. Ahubwo, protocole yacu itanga morphogenesis y'igifu itunguranye, stress ijyanye n'imiterere y'umubiri, biomekanike isa n'impinduka mu mara, uburyo bwo kugera ku bice byigenga bya apical na basolateral, no kongera kurema ibidukikije bito by'ibinyabuzima bya modularity. Kubwibyo, protocole yacu ya morphogenesis ya 3D muri vitro ishobora gutanga uburyo bwuzuzanya bwo gukemura ibibazo by'uburyo busanzwe.
Protocole yacu yibanda ku miterere ya 3D epithelial morphogenesis, aho uturemangingo twa epithelial gusa mu mikurire nta bundi bwoko bw'uturemangingo tuyikikije nka selile za mesenchymal, selile za endothelial, n'uturemangingo tw'ubudahangarwa bw'umubiri. Nkuko byasobanuwe mbere, ishingiro rya protocole yacu ni ukwinjizwa kwa epithelial morphogenesis binyuze mu gukuraho ibibuza morphogen biva ku ruhande rw'inyuma rw'uburyo bwinjiye. Mu gihe modularity ikomeye ya gut-on-a-chip yacu na hybrid-on-a-chip bidufasha kongera kurema urwego rwa 3D epithelial, izindi ngorane z'ibinyabuzima nka epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35, kandi, muri moderi yacu, imiterere ya crypt-villus itanga niches za stem cells muri basal crypts iracyagomba kwitabwaho cyane. Uturemangingo twa stromal (urugero: fibroblasts) muri mesenchyme bigira uruhare runini mu gukora poroteyine za ECM no kugenzura imiterere y'amara muri vivo35,37,38. Kongeramo uturemangingo twa mesenchymal Kuri modeli yacu byongereye uburyo bwo guhuza uturemangingo n'imikorere myiza y'uturemangingo. Urusobe rw'inyuma (ni ukuvuga imiyoboro y'amaraso cyangwa imitsi y'amaraso) rugira uruhare runini mu kugenzura ubwikorezi bw'uturemangingo39 no gushyira uturemangingo tw'ubudahangarwa mu mara. Byongeye kandi, ibice by'imitsi bishobora guhuzwa hagati y'uturemangingo ni ngombwa iyo imiterere y'uturemangingo igenewe kugaragaza imikoranire y'uturemangingo twinshi. Kubwibyo, uturemangingo tw'inyuma dushobora gukenera gushyirwamo kugira ngo tugaragaze imiterere nyayo y'umubiri hamwe n'uburyo ingingo zigaragara. Uturemangingo tw'ubudahangarwa dukomoka ku murwayi na two ni ingenzi mu kugaragaza uburyo ubudahangarwa bw'umubiri busanzwe, imiterere ya antigen, uburyo ubudahangarwa bw'umubiri busanzwe bukoreshwa, n'ubudahangarwa bw'umubiri bwihariye mu rwego rwo kwigana indwara z'amara.
Gukoresha utubumbe duto two mu bwoko bwa "hybrid chips" biroroshye kurusha "gut-on-a-chip" kuko igikoresho cyoroshye kandi gukoresha "Transwell inserts" bituma "epithelium" yo mu mara ikura neza. Ariko, "Transwell inserts" zigurishwa zifite "polyester membranes" ntabwo zikora neza kandi ntizishobora kwigana "peristaltic-like movements". Byongeye kandi, "apical part" ya "Transwell insert" yashyizwe muri "hybrid chip" yagumye ihagaze nta "stress" ku ruhande rw'utubumbe. Birumvikana ko imiterere idahinduka mu tubumbe duto two mu bwoko bwa "apical" idakunze gutuma "bacterial co-culture" iramba mu tubumbe duto two mu bwoko bwa "hybrid chips". Nubwo dushobora gutera "morphogenesis" ya 3D mu tubumbe duto two mu bwoko bwa "Transwell" iyo dukoresha "hybrid chips", kubura kwa "biomekanike" zijyanye n'imikorere y'umubiri n'amazi yo mu bwoko bwa "apical fluid" bishobora kugabanya ubushobozi bwa "hybrid chip platforms" ku buryo bushoboka.
Kuvugurura ku buryo bwuzuye umurongo wa crypt-villus mu nda no mu miterere ya hybrid-on-a-chip ntabwo byagaragajwe neza. Kubera ko imiterere itangirana n'urutonde rw'inyuma, imiterere mito ya 3D ntabwo isobanura imiterere nk'iy'inyuma. Nubwo twagaragaje umubare w'uturemangingo twiyongera hafi y'aho basal crypt domain muri microengineered 3D epithelium, crypt n'uturere twa villous ntibyari byarashyizwe ku murongo neza. Nubwo inzira zo hejuru zo hejuru kuri chip zituma uburebure bwa microengineered epithelium bwiyongera, uburebure ntarengwa buracyari ~ 300–400 µm. Ubujyakuzimu nyabwo bw'inyuma y'amara y'umuntu mu mara mato n'amanini ni ~ 135 µm na ~ 400 µm, uko bikurikirana, kandi uburebure bwa small intestine villi ni ~ 600 µm41.
Mu buryo bwo gufotora, amashusho ya microarchitectures ya 3D ashobora kugarukira ku mara ari kuri chip, kubera ko intera ikenewe yo gukora kuva kuri lens y’icyerekezo kugeza ku gice cya epithelial iri ku rugero rwa milimetero nke. Kugira ngo iki kibazo gikemuke, hashobora kuba hakenewe intego iri kure. Byongeye kandi, gukora ibice bito byo gutegura ingero z’ishusho biragoye bitewe n’uburemere bwinshi bwa PDMS. Byongeye kandi, kubera ko gukora microfabrication y’amara kuri chip bisaba gufatana burundu hagati ya buri gice, biragoye cyane gufungura cyangwa gukuraho igice cyo hejuru kugira ngo usuzume imiterere y’ubuso bw’igice cya epithelial. Urugero, hakoreshejwe microscope ya scanning electron (SEM).
Kuba PDMS idashobora gushonga mu mazi byagize ingaruka mbi ku bushakashatsi bushingiye kuri microfluidic bukorwa ku ndunduro nto zidafite amazi, kubera ko PDMS ishobora kwinjiza izo ndunduro zidafite amazi mu buryo bwihariye. Uburyo bwo guhindura PDMS bushobora kwitabwaho n'ibindi bikoresho bya polymer. Ubundi buryo, guhindura ubuso bwa PDMS (urugero, gusiga ibikoresho bya lipophilic 42 cyangwa poly (ethylene glycol) 43) bishobora gufatwa nk'ikintu kigabanya kwinjizwa kwa ndunduro zidafite amazi.
Amaherezo, uburyo bwacu ntibwarasobanuwe neza mu gutanga uburyo bwo gusuzuma ibintu byinshi cyangwa urubuga rworoshye rwo gukora igerageza "rukwiriye bose". Porogaramu iriho ubu isaba pompe ya siringi kuri buri gikoresho, ifata umwanya muri couverture ya CO2 kandi ikabuza igerageza rinini. Iyi mbogamizi ishobora kunozwa cyane bitewe n'uburyo bushya bwo gukwirakwiza imiterere y'imiti (urugero: 24-well, 96-well, cyangwa 384-wells inserts zituma habaho kongera no gukuraho imiyoboro y'amazi isanzwe).
Kugira ngo dutere imiterere ya 3D ya epithelium y'amara y'umuntu muri vitro, twakoresheje igikoresho cy'amara cy'imashini ntoya irimo imiyoboro ibiri ihwanye hamwe n'urukiramende rworoshye ruri hagati kugira ngo dukore ihuriro rya lumen-capillary. Tugaragaza kandi ikoreshwa ry'igikoresho cy'imashini ntoya ifite umuyoboro umwe (imashini ntoya) itanga urujya n'uruza rw'imbere munsi y'ibice bya epithelial byashyizwe ku biti bya Transwell. Muri izo mbuga zombi, imiterere y'uturemangingo dutandukanye tw'amara y'umuntu ishobora kugaragazwa hakoreshejwe uburyo bwo guhindura icyerekezo cy'urujya n'uruza kugira ngo dukureho antagonists za morphogen mu gice cy'amara y'imbere. Uburyo bwose bwo kugerageza (Ishusho ya 1) bugizwe n'ibice bitanu: (i) gukora microfabrication ya intestine ya intestine cyangwa Transwell insertable hybrid chip (intambwe ya 1-5; Agasanduku ka 1), (ii) gutegura uturemangingo tw'amara y'intanga (uturemangingo twa Caco-2) cyangwa organoids z'amara y'umuntu; udusanduku twa 2-5), (iii) ubuhinzi bw'uturemangingo tw'inda ku turemangingo tw'inda cyangwa uturemangingo tw'imvange (intambwe ya 6-9), (iv) kwinjizwa kwa morphogenesis ya 3D muri vitro (intambwe ya 10) na (v)) kugira ngo hamenyekane imiterere mito ya 3D epithelial (intambwe ya 11-24). Amaherezo, itsinda rikwiye ryo kugenzura (ryavuzwe haruguru) ryagenewe kwemeza akamaro ka morphogenesis ya in vitro hagereranywa morphogenesis ya epithelial n'uburyo bwo kugenzura imiterere y'ahantu, ubw'igihe, ubw'ibikenewe, cyangwa uburyo bwo kugenzura.
Twakoresheje uburyo bubiri butandukanye bwo gutandukanya imiterere y’ibinyabuzima: gut-on-a-chip ifite imiyoboro igororotse cyangwa imiyoboro idahuza, cyangwa utubumbe duvanze turimo Transwell (TW) mu gikoresho cy’amazi make, cyakozwe nk'uko byasobanuwe mu gasanduku ka 1, n'intambwe ya 1 -5. "Gukora Ibikoresho" bigaragaza intambwe z'ingenzi mu gukora utubumbe duto cyangwa utubumbe duvanze." Umuco w'uturemangingo tw’inda z’abantu" usobanura isoko y'uturemangingo (Caco-2 cyangwa organoids z’inda z’abantu) n'uburyo bwo gutandukanya imiterere y’ibinyabuzima bwakoreshejwe muri iyi protocole. "In vitro morphogenesis" yerekana intambwe rusange aho uturemangingo tw’inda z’abantu dukomoka kuri Caco-2 cyangwa organoids duhingwa ku tubumbe duto cyangwa ku tubumbe duto twa Transwell, hagakurikiraho kwinjizwa kwa morphogenesis ya 3D no gushyiraho imiterere y’utubumbe. Nimero y’intambwe ya gahunda cyangwa inomero y’agasanduku igaragara munsi ya buri mwambi. Porogaramu itanga ingero z'uburyo urwego rw’utubumbe tw’inda rwashyizweho rushobora gukoreshwa, urugero, mu kugena itandukaniro ry’uturemangingo, inyigo z’imiterere y’inda, gushyiraho ibidukikije by’uturemangingo tw’ibinyabuzima, n’indwara. Ishusho. Amashusho y'ubudahangarwa bw'umubiri muri "Cell Differentiation" agaragaza nucleus, F-actin na MUC2 bigaragazwa mu gice cya 3D Caco-2 epithelial cyakozwe ku kirahure cy'igifu. Ibimenyetso bya MUC2 biri mu tunyangingo tw'ibitunguru n'urumogi ruturuka ku buso bw'urumogi. Amashusho y'ubudahangarwa bw'umubiri muri Gut Physiology agaragaza urumogi rukorwa no gusiga irangi kuri aside sialic na N-acetylglucosamine residues hakoreshejwe fluorescent wheat germ agglutinin. Amashusho abiri ahuye muri "Host-Microbe Co-Cultures" agaragaza imiterere y'utunyangingo tw'umubiri mu mara kuri chip. Igice cy'ibumoso kigaragaza imiterere y'utunyangingo twa E. coli fluorescent protein (GFP) hamwe n'utunyangingo twa 3D Caco-2 epithelial. Igice cy'iburyo kigaragaza aho GFP E. coli ihingwa hamwe n'utunyangingo twa 3D Caco-2 epithelial, hagakurikiraho irangi rya immunofluorescence hamwe na F-actin (umutuku) na nuclei (ubururu). Ishusho y'indwara igaragaza amara meza ugereranyije n'atose mu mara Udukoko tw’ububyimbirwe turi mu kaga k’umubiri hamwe na antigen za bagiteri (urugero, lipopolysaccharide, LPS) n’uturemangingo tw’ubudahangarwa bw’umubiri (urugero, PBMC; icyatsi). Uturemangingo twa Caco-2 twahinzwe kugira ngo habeho urwego rwa 3D epithelial. Umugozi w’ingero, 50 µm. Amashusho ari ku murongo wo hasi: "Itandukaniro ry’uturemangingo" ryahinduwe ku ruhushya ruturutse ku nyandiko. 2. Oxford University Press; Byasubiwemo ku ruhushya ruturutse ku nyandiko. 5. NAS; "Host-Microbe Co-Culture" yahinduwe ku ruhushya ruturutse ku nyandiko. 3. NAS; "Disease Modeling" yahinduwe ku ruhushya ruturutse ku nyandiko. 5. NAS.
Uduce twombi two mu nda n'utwo mu bwoko bwa hybrid twakozwe hakoreshejwe kopi za PDMS zakuwe mu turemangingo twa silikoni hakoreshejwe uburyo bworoshye bwa lithography1,44 kandi zishushanyije na SU-8. Imiterere y'imiyoboro mito muri buri chip igenwa no gusuzuma hydrodynamics nka shear stress n'umuvuduko wa hydrodynamic1,4,12. Igishushanyo cya mbere cya gut-on-a-chip (Extended Data Fig. 1a), cyari kigizwe n'imiyoboro ibiri igororotse ihuriweho, cyahindutse complex gut-on-a-chip (Extended Data Fig. 1b) irimo imiyoboro ibiri igoramye kugira ngo itume igihe cyo guturamo amazi cyiyongera, imiterere y'urujya n'uruza rw'uturemangingo, no guhinduka kwa multiaxial kw'uturemangingo twakuriyemo (Ishusho ya 2a–f) 12. Iyo biomekanike y'uturemangingo duto two mu nda ikeneye kuvugururwa, utwo duce duto two mu nda dushobora gutoranywa. Twagaragaje ko Gut-Chip ihindagurika inatera cyane morphogenesis ya 3D mu gihe kimwe hamwe n'urugero rumwe rw'ikura ry'impiswi ugereranije n'urw'umwimerere. Gut-Chip, hatitawe ku bwoko bw'uturemangingo twakuwemo. Kubwibyo, kugira ngo habeho imiterere ya 3D, imiterere y'uturemangingo tw'intanga ngabo iri ku murongo n'iy'intanga ngabo irahinduka. Amafoto ya PDMS yavuwe ku bimera bya silikoni hamwe n'imiterere ya SU-8 yatanze imiterere mibi nyuma yo gusenyuka (Ishusho ya 2a). Kugira ngo hakorwe amara kuri chip, urwego rwo hejuru rwa PDMS rwateguwe rwahujwe ku buryo bukurikiranye kuri filime ya PDMS ifite imyenge hanyuma ruhuzwa n'urwego rwo hasi rwa PDMS binyuze mu guhuza bidasubirwaho hakoreshejwe icyuma gitanga umuti wa corona (Ishusho ya 2b–f). Kugira ngo hakorwe uturemangingo tw'intanga ngabo, amafoto ya PDMS yavuwe yahujwe ku birahuri kugira ngo hakorwe ibikoresho bya microfluidic by'umuyoboro umwe bishobora kwakira Transwell inserts (Ishusho ya 2h n'Ishusho ya Extended Data Fig. 2). Uburyo bwo guhuza bukorwa havurwa ubuso bwa PDMS replica n'ikirahure hakoreshejwe plasma ya ogisijeni cyangwa ubuvuzi bwa corona. Nyuma yo gusukura igikoresho cya microfabricated gifatanye n'umuyoboro wa silikoni, igikoresho cyari cyiteguye gukora imiterere ya 3D ya epithelium y'amara (Ishusho ya 2g).
a, Ishusho y'icyitegererezo cy'itegurwa ry'ibice bya PDMS bivuye mu mabumba ya silikoni afite ishusho ya SU-8. Umuti wa PDMS utaravurwa wasutswe ku ibumba rya silikoni (ibumoso), wavuwe kuri 60 °C (hagati) hanyuma uvanwaho (iburyo). PDMS yaciwemo ibice irasukurwa kugira ngo ikomeze gukoreshwa.b, Ifoto y'ibumba rya silikoni ryakoreshejwe mu gutegura urwego rwo hejuru rwa PDMS.c, Ifoto y'ibumba rya silikoni ryakoreshejwe mu gukora membrane ifite imyenge ya PDMS.d, Urukurikirane rw'amafoto y'ibice byo hejuru n'ibyo hasi bya PDMS n'igikoresho cy'amara cyateranijwe kuri chip.e., Ishusho y'uburyo ibice byo hejuru, membrane, n'ibyo hasi bya PDMS bihujwe. Buri cyiciro gihuzwa mu buryo budasubirwaho n'ubuvuzi bwa plasma cyangwa corona.f, Ishusho y'igikoresho cyakozwe mu gut-on-a-chip gifite imiyoboro minini yashyizwemo hamwe n'ibyumba by'ubushyuhe.g, Gushyiraho gut-on-a-chip kugira ngo haboneke imiterere y'uturemangingo twa microfluidic. Inda yakozwe ku ibumba yateranijwe n'umuyoboro wa silikoni na syringe byashyizwe ku gapfundikizo. Igikoresho cya chip cyashyizwe ku gipfundikizo cya Isahani ya Petri ya mm 150 yo gutunganya. Igikoresho gikoreshwa mu gufunga umuyoboro wa silicone.h, Amashusho yerekana uko ikorwa rya chips za hybride n'imiterere ya 3D hakoreshejwe chips za hybride. Ibyinjizwa bya Transwell byateguwe ku giti cyabyo kugira ngo bivumburwe. Uduce twa 2D tw'uturemangingo twa epithelial y'amara twashyizwe muri chips za hybride kugira ngo bitere imiterere ya 3D y'amara. Utu duce duto dushyirwa mu miyoboro mito munsi y'urwego rw'uturemangingo rwashyizweho kuri Transwell. Uduce twa scale, cm 1.h Byasubiwemo uruhushya ruturutse ku nyandiko.4. Elsevier.
Muri iyi protocole, umurongo w’uturemangingo twa Caco-2 n’uturemangingo tw’intanga byakoreshejwe nk’isoko ry’uturemangingo (Ishusho ya 3a). Ubwoko bwombi bw’uturemangingo bwatewe ku giti cyabwo (Agasanduku ka 2 n’Agasanduku ka 5) kandi bwakoreshejwe mu gutera imiyoboro mito ya ECM y’uturemangingo tw’intanga cyangwa Transwell. Iyo uturemangingo duhuye (>95% by’ubwinshi bw’uturemangingo mu macupa), uturemangingo twa Caco-2 twatewe ku gihe (hagati y’inzira ya 10 na 50) mu ducupa twa T dusarurwa kugira ngo dutegure uduce twakuwemo uturemangingo hakoreshejwe amazi ya trypsinize (agasanduku ka 2). Uturemangingo tw’intanga tw’abantu twakuwe mu mara cyangwa mu kubagwa twatewe mu duce twa Matrigel scaffold domes mu masahani 24 kugira ngo dushyigikire imiterere y’ibidukikije. Uturemangingo turimo ibintu by’ingenzi (nka Wnt, R-spondin, na Noggin) kandi ibintu bikura byateguwe nk’uko byasobanuwe mu Gasanduku ka 3 byongerwagaho buri munsi kugeza ubwo uturemangingo twakuze tukagera kuri ~ 500 µm mu murambararo. Uturemangingo twakuze neza turasarurwa kandi Bitandukanyijwe mu turemangingo duto kugira ngo bitere ku mara cyangwa Transwell inserts kuri chip (Agasanduku ka 5). Nkuko twabitangaje mbere, bishobora gutandukanywa hakurikijwe ubwoko bw'indwara12,13 (urugero: indwara ya kanseri y'ibisebe, indwara ya Crohn, kanseri y'inkondo y'umura, cyangwa umutanga usanzwe), aho ibisebe biherereye (urugero: ahantu hakomeretse ugereranyije n'aho hadakomeretse) n'aho igifu giherereye mu nzira (urugero: duodenum, jejunum, ileum, cecum, colon, cyangwa rectum). Dutanga uburyo bwiza bwo gupima organoids zo mu mara (coloids) zisanzwe zikenera ubwinshi bwa morphogens kurusha organoids zo mu mara mato.
a, Imikorere yo kwinjiza imiterere y’igifu mu kirahure cy’igifu. Ingirabuzimafatizo z’amara ya Caco-2 n’ingirabuzimafatizo z’igifu byakoreshejwe muri iyi porotokoli kugira ngo hagaragazwe imiterere ya 3D. Uturemangingo tw’ingirabuzimafatizo twashyizwe mu gikoresho cyateguwe cyo mu nda (gutegura ibyuma). Iyo uturemangingo tumaze guterwa (guterwa) kandi tugashyirwa ku ruhu rw’imyenge rwa PDMS ku munsi wa 0 (D0), urujya n’uruza rw’ingirabuzimafatizo (AP) ruratangira kandi rugakomeza gukoreshwa mu minsi 2 ya mbere (urujya n’uruza, AP, D0-D2). Urujya n’uruza rw’ingirabuzimafatizo (BL) narwo rutangira hamwe n’ingendo zo kwagura (kurambura, gutembera, AP na BL) iyo habayeho urwego rwuzuye rwa 2D. Urujya n’uruza rw’amara rwa 3D rwabayeho ku buryo butunguranye nyuma y’iminsi 5 y’imikorobe y’amazi (morphogenesis, D5). Amashusho y’itandukaniro ry’icyiciro agaragaza imiterere y’uturemangingo twa Caco-2 kuri buri ntambwe cyangwa igihe cy’igerageza (imbonerahamwe y’umurongo, 100 µm). Ibishushanyo bine byerekana imiyoboro ijyanye nayo y’imiterere y’igifu (hejuru iburyo). Utwambi duto turi muri shaki tugaragaza icyerekezo cy’amazi atembera.b, Ishusho ya SEM igaragaza ubuso bw’inyuma bwa epithelium ya 3D Caco-2 (ibumoso). Agace kerekana agace kanini (agasanduku k’umweru gafite shaki) kerekana microvilli yongeye kuvugururwa ku rwego rwa 3D Caco-2 (iburyo).c, Ishusho y’imbere itambitse ya Caco-2 3D yashyizweho, claudin (ZO-1, umutuku) n’uturemangingo tw’umupaka duhoraho twanditseho F-actin (icyatsi) na nuclei (ubururu) Ishusho ya immunofluorescence confocal y’uturemangingo tw’inyuma ku turemangingo tw’amara. Utwambi twerekana shaki yo hagati tugaragaza aho urwego rw’ibanze ruri kuri buri shaki.d, Igihe cy’impinduka mu miterere ya organoids yatewe kuri chip yabonetse hakoreshejwe mikorosikopi ya phase contrast ku munsi wa 3, 7, 9, 11, na 13. Agace k’imbere (hejuru iburyo) kerekana ubwinshi bw’ishusho yatanzwe.e., DIC photomicrograph ya organoid 3D epithelium yashyizweho mu mara ku gice cyafashwe ku munsi wa 7.f, Amashusho y’ubudahangarwa bw’umubiri agaragaza ibimenyetso by’uturemangingo fatizo (LGR5; magenta), uturemangingo tw’indobo (MUC2; icyatsi), F-actin (imvi) na nuclei (cyan) byakuriye ku turemangingo tw’igifu mu gihe cy’iminsi 3, (ibumoso) na organoide z’iminsi 13 (hagati) ku gice cy’inyuma. Reba kandi Ishusho ya 3 y’amakuru yagutse, igaragaza ibimenyetso bya LGR5 nta bimenyetso bya MUC2. Amashusho y’ubudahangarwa agaragaza imiterere y’inyuma (iburyo) ya epithelium ya organoid ya 3D yashyizwe mu mara ku gice cy’inyuma binyuze mu gusiga irangi rya plasma hakoreshejwe CellMask (iburyo) ku munsi wa 13 w’ivumbura. Umwanya w’ubunini ni 50 μm keretse iyo byavuzwe ukundi.b Byasubiwemo uruhushya ruvuye ku ivumbura.2. Oxford University Press; c Byahinduwe uruhushya ruvuye ku ivumbura.2. Oxford University Press; e na f byahinduwe uruhushya ruvuye ku ivumbura.12 Bikurikije Creative Commons License CC BY 4.0.
Mu mara ku gipfundikizo, ni ngombwa guhindura ubuso buto ... epithelium kugira ngo itere imiterere ya 3D ku buso bwa PDMS.
Guteranya udukoko two mu bwoko bwa Transwell nabyo bisaba ko dushyirwamo ECM mbere yo gutera imbuto; icyakora, guteranya Transwell ntibisaba ingamba zigoye zo kuvura mbere yo gukoresha kugira ngo hakoreshwe ubuso bw'uturemangingo dufite imyenge. Ku bijyanye no gukura uturemangingo twa Caco-2 ku turemangingo twa Transwell, guteranya ECM ku turemangingo dufite imyenge byihutisha gufatanya kwa uturemangingo twa Caco-2 twatandukanye (
Imiterere y’imiterere y’ibice bya epithelial bya 3D bya microengineered ishobora gusesengurwa hakoreshejwe uburyo butandukanye bwo gufata amashusho, harimo microscopy ya phase contrast, microscopy ya differential interference contrast (DIC), SEM, cyangwa microscopy ya confocal ya immunofluorescence (Ishusho ya 3 na 4). Phase contrast cyangwa DIC imaging ishobora gukorwa byoroshye igihe icyo ari cyo cyose mu gihe cyo gukuriramo kugira ngo ikurikirane imiterere n’isohoka ry’ibice bya epithelial bya 3D. Bitewe n’ubuhanga bw’izuba bwa PDMS na polyester films, platforms za gut-on-a-chip na hybrid chip zishobora gutanga amashusho y’igihe nyacyo nta mpamvu yo gutandukanya cyangwa gukuraho igikoresho. Mu gihe cyo gukora imaging ya immunofluorescence (Ishusho ya 1, 3c, f na 4b, c), uturemangingo dusanzwe dufatana na 4% (wt/vol) paraformaldehyde (PFA), hagakurikiraho Triton X-100 na 2% (wt/vol)) bovine serum albumin (BSA), mu buryo bukurikiranye. Bitewe n’ubwoko bw’uturemangingo, ibintu bitandukanye bihinduka, Ibikoresho bipima umwuka, n'ibintu bifunga bishobora gukoreshwa. Udukoko tw'ibanze twibasira uturemangingo cyangwa ibimenyetso by'akarere bikoreshwa mu kugaragaza uturemangingo tudakora neza turi kuri chip, hagakurikiraho uturemangingo tw'inyongera hamwe n'irangi rirwanya ibara ryibasira nucleus (urugero, 4′, 6-diamidino-2-phenylene) indole, DAPI) cyangwa F-actin (urugero, phalloidin yanditseho fluorescent). Ishusho ishingiye kuri fluorescence ishobora kandi gukorerwa muri situ kugira ngo hamenyekane umusaruro w'uturemangingo (Ishusho ya 1, "Gutandukanya uturemangingo" na "Uburyo bw'uturemangingo tw'imibiri"), kwibasira uturemangingo tw'imibiri mu buryo butunguranye (Ishusho ya 1, "Guhuza uturemangingo tw'umurwayi"), kwibasira uturemangingo tw'ubudahangarwa (Ishusho ya 1, 'Gushushanya indwara') cyangwa imiterere y'imiterere ya 3D epithelial (Ishusho ya 3c, f na 4b, c). Mu gihe uhindura amara kuri chip kugira ngo utandukanye urwego rwo hejuru n'urwego rwo hasi rwa microchannel, nk'uko bigaragazwa ku Ishusho ya 2, epithelial ya 3D Imiterere y'uturemangingo ndetse na microvilli ku mupaka w'uburoso bw'inyuma bishobora kugaragazwa na SEM (Ishusho ya 3b). Imiterere y'ibimenyetso by'itandukaniro ishobora gupimwa hakoreshejwe uburyo bwa PCR5 cyangwa se RNA y'akaremangingo kamwe. Muri iki gihe, urwego rwa 3D rw'uturemangingo tw'inyuma twakuriye mu turemangingo tw'igifu cyangwa uturemangingo tw'imvange bishakwa hakoreshejwe trypsinisation hanyuma bigakoreshwa mu isesengura rya molekile cyangwa genetiki.
a, Imikorere yo gutangiza imiterere y'amara mu gice cy'uruvange rw'inyabutabire. Caco-2 na organoids zo mu nda bikoreshwa muri iyi porotoli kugira ngo byerekane imiterere ya 3D mu gice cy'uruvange rw'inyabutabire. Uturemangingo tw'inyabutabire twitandukanyije twatewe mu bice bya Transwell byateguwe (TW prep; reba ishusho iri hepfo). Uturemangingo tumaze guterwa (guterwa) no gufatanywa n'uturemangingo twa polyester mu bice bya Transwell, uturemangingo twose twatewe mu bihe bitahinduka (TW culture). Nyuma y'iminsi 7, igice kimwe cya Transwell kirimo urwego rumwe rwa 2D rw'uturemangingo tw'inyabutabire cyashyizwe mu gice cy'uruvange rw'inyabutabire kugira ngo hatangwe uruvange rw'inyabutabire (Flow, BL), amaherezo byatumye habaho urwego rwa 3D rw'inyabutabire (morphogenesis). Imiterere y'itandukaniro ry'icyiciro igaragaza imiterere y'uturemangingo tw'inyabutabire tw'umubiri w'umuntu twakuwe ku gice gisanzwe cy'inyabutabire (umurongo wa C103) kiri kuzamuka kuri buri ntambwe cyangwa igihe cy'igerageza. Imiterere y'inyabutabire zo hejuru igaragaza imiterere y'igerageza kuri buri ntambwe.b, Uturemangingo tw'inyabutabire (igishushanyo mbonera cy'ibumoso) dushobora gutuma habaho imiterere ya 3D y'inyabutabire y'inyabutabire y'inyabutabire y'inyabutabire Uturemangingo dufite imiterere ya microscopy yo hejuru-hasi yafashwe ku myanya itandukanye ya Z (hejuru, hagati, no hepfo; reba imirongo y'iburyo n'utudomo bihuye). byagaragaje imiterere igaragara. F-actin (cyan), nucleus (imvi).c, Fluorescence confocal micrographs (ishusho ya 3D) y'uturemangingo tw'inyuma dukomoka kuri organoide twakuriye muri Transwell itaziguye (TW; ishusho iri mu gasanduku k'umweru gafite dashe) ugereranije na chip hybrid (ishusho nini cyane) ugereranije imiterere ya 2D ugereranyije na 3D, uko bikurikirana. Uturemangingo tubiri tw'imiterere ya 2D ihagaze (ishusho iri mu mfuruka yo hejuru iburyo; "XZ") na two tugaragaza imiterere ya 2D na 3D. Uduce tw'ingero, 100 µm.c Byacapwe kandi byemejwe n'inyigo.4. Elsevier.
Uburyo bwo kugenzura bushobora gutegurwa binyuze mu guteranya uturemangingo tumwe (Caco-2 cyangwa uturemangingo tw’ingirabuzimafatizo tw’inkondo y’umura) mu turemangingo tw’ibice bibiri mu buryo busanzwe bwo kwita ku miterere y’umubiri. Ikigaragara ni uko kugabanuka kw’intungamubiri bishobora guterwa n’ubushobozi buke bw’imiyoboro mito (ni ukuvuga ~ 4 µL mu muyoboro wo hejuru ku gishushanyo cy’umwimerere cy’uturemangingo tw’imbere). Kubwibyo, imiterere y’uturemangingo mbere na nyuma yo gukoresha umuyoboro w’imbere w’ingirabuzimafatizo nayo ishobora kugereranywa.
Uburyo bwo gushushanya bugomba gukorerwa mu cyumba gisukuye. Kuri buri cyiciro kiri kuri chip (urwego rwo hejuru n'urwo hasi n'ururabo) na chips za hybrid, hakoreshejwe photomask zitandukanye kandi zikorwa ku dupapuro twa silikoni dutandukanye kuko uburebure bw'imiyoboro mito bwari butandukanye. Uburebure bw'imiyoboro mito yo hejuru n'iyo hasi y'igifu kuri chip ni 500 µm na 200 µm, uko bikurikirana. Uburebure bw'imiyoboro mito ya hybrid ni 200 µm.
Shyira wafer ya silikoni ya santimetero 3 mu isahani irimo acetone. Zungurutsa isahani witonze mu gihe cy'amasegonda 30, hanyuma wumishe wafer mu mwuka. Shyira wafer ku isahani ifite IPA, hanyuma uzunguruke isahani mu gihe cy'iminota 30 kugira ngo isukure.
Umuti wa piranha (uvanze na peroxide ya hydrogen na aside sulfurike ikungahaye kuri 1:3 (vol/vol)) ushobora gukoreshwa mu buryo bwihariye kugira ngo ibisigazwa by’ibinyabuzima bikurwe ku buso bwa silicon wafer.
Umuti wa Piranha urimo kwangirika cyane kandi utanga ubushyuhe. Hakenewe ingamba zindi z'umutekano. Kugira ngo imyanda ikonje, reka umuti ukonje hanyuma uwushyire mu gikoresho cy'imyanda gisukuye kandi cyumye. Koresha ibikoresho bya kabiri kandi ushyireho ikimenyetso gikwiye ku bikoresho by'imyanda. Nyamuneka kurikiza amabwiriza y'umutekano y'ikigo kugira ngo ubone uburyo burambuye bwo gukora.
Shyira amazi mu duce twa wafers ku isahani ishyushye ya 200 °C mu gihe cy'iminota 10. Nyuma yo kubura amazi, wafer yanyeganyejwe inshuro eshanu mu kirere kugira ngo ikonje.
Suka garama 10 za photoresist SU-8 2100 hagati muri wafer ya silikoni isukuye. Koresha utubuto kugira ngo ushyire photoresist kuri wafer neza. Rimwe na rimwe shyira wafer ku isahani ishyushye ya 65°C kugira ngo photoresist idafata neza kandi yorohe kuyikwirakwiza. Ntugashyire wafer ku isahani ishyushye.
SU-8 yakwirakwijwe ku buryo bungana kuri wafer hakoreshejwe irangi rya spin. Tegura uburyo SU-8 izenguruka mu gihe cy'amasegonda 5-10 kugira ngo ikwirakwizwe kuri 500 rpm ku muvuduko wa 100 rpm/s. Shyiraho umuvuduko w'ingenzi ku bugari bwa 200 µm kuri 1,500 rpm, cyangwa ugere ku bugari bwa 250 µm (bigatuma uburebure bwa 500 µm ku gice cyo hejuru cy'igifu kiri kuri chip; reba "Intambwe z'ingenzi" hepfo) ishyirwa ku muvuduko wa 300 rpm/s amasegonda 30 kuri 1,200 rpm.
Umuvuduko w'ingenzi wo kuzunguruka ushobora guhindurwa hakurikijwe ubunini bw'icyitegererezo cya SU-8 kuri wafer ya silikoni.
Kugira ngo hakorwe imiterere ya SU-8 ifite uburebure bwa µm 500 ku gice cyo hejuru cy'igifu kiri ku gipfundikizo, irangi rya spin n'intambwe zo guteka byoroshye by'iyi Sanduku (intambwe ya 7 na 8) byasubiwemo bikurikirana (reba intambwe ya 9) kugira ngo hakorwe urwego rubiri rwa µm 250. Urwego runini rwa SU-8, rushobora gushyirwaho urwego no guhuzwa n'imirasire ya UV mu ntambwe ya 12 y'iyi Sanduku, bigatuma urwego rugira uburebure bwa µm 500.
Teka neza wafers za SU-8 ushyira wafers ku isahani ishyushye kuri 65 °C mu gihe cy'iminota 5, hanyuma uhindure imiterere kuri 95 °C hanyuma uzishyire mu gihe cy'indi minota 40.
Kugira ngo ugere ku burebure bwa μm 500 bw'igishushanyo cya SU-8 mu gice cyo hejuru cy'umuyoboro muto, subiramo intambwe ya 7 na 8 kugira ngo ukore ibice bibiri bya SU-8 bifite ubugari bwa μm 250.
Ukoresheje UV Mask Aligner, kora ikizamini cy'itara ukurikije amabwiriza y'uwakoze kugira ngo ubare igihe cyo gushyiramo wafer. (igihe cyo gushyiramo, ms) = (igipimo cyo gushyiramo, mJ/cm2)/(ingufu z'itara, mW/cm2).
Nyuma yo kumenya igihe cyo gushyiramo aka gapfukamunwa, shyira agapfukamunwa ku gikoresho cyo gufata agapfukamunwa ka UV mask aligner hanyuma ushyire agapfukamunwa ku gapfukamunwa gatwikiriwe na SU-8.
Shyira ubuso bwacapwe bwa photomask ku ruhande rwa SU-8 rwa silicon wafer kugira ngo ugabanye ikwirakwira rya UV.
Shyira wafer ya SU-8 hamwe na photomask ihagaze kugeza kuri 260 mJ/cm2 y'urumuri rwa UV mu gihe cyagenwe cyo kwigaragaza (reba intambwe ya 10 y'aka gasanduku).
Nyuma yo gushyuha hakoreshejwe imirasire ya UV, utugati twa silikoni twa SU-8 twatekeshejwe kuri 65°C mu gihe cy'iminota 5 na 95°C mu gihe cy'iminota 15 kuri buri sahani ishyushye kugira ngo hakorwe imiterere ifite uburebure bwa 200 μm. Ongera igihe cyo guteka nyuma yo guteka kuri 95°C kugeza ku minota 30 kugira ngo hakorwe imiterere ifite uburebure bwa 500 μm.
Igikoresho gisukwa mu isahani y'ikirahure, hanyuma agace gatetse gashyirwa mu isahani. Ingano y'igikoresho cya SU-8 ishobora gutandukana bitewe n'ingano y'isahani y'ikirahure. Menya neza ko wakoresheje igikoresho gihagije cya SU-8 kugira ngo ukureho burundu SU-8 itaragaragara. Urugero, iyo ukoresha isahani y'ikirahure ifite umurambararo wa mm 150 ifite ubushobozi bwa litiro 1, koresha ~ mL 300 za gikoresho cya SU-8. Kora ifumbire mu gihe cy'iminota 25 uhinduranya buhoro buhoro rimwe na rimwe.
Sukura ifu yavumbuwe ukoresheje ~ 10 mL y'umusaruro mushya hanyuma ushyiremo IPA ukoresheje umuti ukoresheje pipette.
Shyira wafer mu cyuma gisukura plasma hanyuma ushyire plasma ya ogisijeni (umwuka wo mu kirere, igitutu cya 1 × 10−5 Torr, ingufu za 125 W) mu gihe cy'iminota 1.5.
Shyira wafer mu cyuma gipima umwuka urimo agace k'ikirahure imbere. Wafer n'uduce dushobora gushyirwa iruhande. Niba icyuma gipima umwuka urimo umwuka kigabanyijemo ibice byinshi n'isahani, shyira uduce mu cyumba cyo hasi n'uduce two hejuru. Suka 100 μL y'umuti wa trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane ku gikoresho cy'ikirahure hanyuma ushyiremo vacuum kugira ngo silanization iboneke.
Shyira agacupa k'uturemangingo twa Caco-2 twakonjeshejwe mu bwogero bw'amazi burimo ubushyuhe bwa dogere selisiyusi 37, hanyuma wimurire uturemangingo twashongeshejwe mu icupa rya T75 ririmo mL 15 za dogere selisiyusi 37 za Caco-2 zishyushye mbere.
Kugira ngo uturemangingo twa Caco-2 tunyure mu mazi ashyushye cyane, banza ushyiremo Caco-2 medium ishyushye, PBS, na 0.25% trypsin / 1 mM EDTA mu bwogero bw'amazi bushyushye bwa 37°C.
Sukura umwuka ukoresheje vacuum aspiration. Sukura uturemangingo kabiri ukoresheje 5 mL ya PBS ishyushye usubiramo vacuum aspiration hanyuma wongeremo PBS nshya.
Igihe cyo kohereza: Nyakanga-16-2022
